Cell behavior is regulated through transient activation of protein activities at specific subcellular locations. Our ability to study translocation of proteins has been greatly enhanced by advances in the microscopy of fluorescent protein analogues within living cells. However, in many cases, localized protein activities are controlled not by translocating proteins to the site of action, but by localized activation of a small portion of the protein pool. Hahn, K.; Toutchkine, A. Curr. Opin. Cell Biol. 2002, 14, 167-172; Wouters, F. S.; Verveer, P. J.; Bastiaens, P. I. Trends Cell Biol. 2001, 11, 203-211. Such behaviors are not apparent when studying protein translocations or when using in vitro biochemical approaches. Furthermore, the outcome of signaling protein activation can depend on subtle variations in activation kinetics that are not discernible in the population averages generated by biochemical techniques. For precise quantification of rapid activation kinetics and of the level of protein activation, it is also necessary to measure protein activity in living cells. Wouters, F. S.; Verveer, P. J.; Bastiaens, P. I. Trends Cell Biol. 2001, 11, 203-211; Williams, D. A.; Fogarty, K. E.; Tsien, R. Y.; Fay, F. S. Nature 1985, 318, 558-561; Berridge, M. J. J. Biol. Chem. 1990, 265, 9583-9586.
Protein activity in living cells has occasionally been observed using FRET (fluorescence resonance energy transfer). Similarly, the interactions between two proteins have been observed by tagging each with different fluorophores that undergo FRET when the proteins associate. FRET biosensors have also been built, which bind to a protein only when it adopts a specific conformation. These approaches can be useful, but FRET-based techniques suffer from limitations that prevent the study of many important targets. Proteins undergoing conformational changes often cannot be “sampled” by a biosensor because the protein is bound to a competing ligand or is incorporated in a multi-protein complex, where it is blocked from biosensor access. However, it is precisely such large, unstable complexes that are difficult to reproduce in vitro and whose transient formation in specific locations must be studied in intact cells. Even when a protein is not sterically blocked, derivatization with a fluorophore near regions of conformational change for FRET can affect biological activity. Finally, because FRET is generated through indirect excitation, it produces a relatively weak fluorescence signal. Such a low signal leads to low sensitivity and to the need for complicated methods to differentiate the real signal from autofluorescence or fluorescence of the FRET donor.
Therefore, a need exists for tools that can do more than monitor protein movements, and do so without the abovementioned disadvantages of FRET. There is a need for biosensors that can be used to detect and quantify diverse protein activities, including changing subcellular locations, conformational changes, activation states, posttranslational modifications, and/or small ligand binding of proteins in vivo.